Comparative analysis of Streptococcus suis genomes identifies novel candidate virulence-associated genes in North American isolates.

Streptococcus suis is a significant economic and welfare concern in the swine industry. Pan-genome analysis provides an in-silico approach for the discovery of genes involved in pathogenesis in bacterial pathogens. In this study, we performed pan-genome analysis of 208 S. suis isolates classified into the pathogenic, possibly opportunistic, and commensal pathotypes to identify novel candidate virulence-associated genes (VAGs) of S. suis. Using chi-square tests and LASSO regression models, three accessory pan-genes corresponding to S. suis strain P1/7 markers SSU_RS09525, SSU_RS09155, and SSU_RS03100 (>95% identity) were identified as having a significant association with the pathogenic pathotype. The proposed novel SSU_RS09525 + /SSU_RS09155 + /SSU_RS03100 + genotype identified 96% of the pathogenic pathotype strains, suggesting a novel genotyping scheme for predicting the pathogenicity of S. suis isolates in North America. In addition, mobile genetic elements carrying antimicrobial resistance genes (ARGs) and VAGs were identified but did not appear to play a major role in the spread of ARGs and VAGs.

Serotype and Genotype (Multilocus Sequence Type) of Streptococcus suis Isolates from the United States Serve as Predictors of Pathotype.

Streptococcus suis is a significant cause of mortality in piglets and growing pigs worldwide. The species contains pathogenic and commensal strains, with pathogenic strains causing meningitis, arthritis, endocarditis, polyserositis, and septicemia. Serotyping and multilocus sequence typing (MLST) are primary methods to differentiate strains, but the information is limited for strains found in the United States. The objective of this study was to characterize the diversity of 208 S. suis isolates collected between 2014 and 2017 across North America (mainly the United States) by serotyping and MLST and to investigate associations between subtype and pathotype classifications (pathogenic, possibly opportunistic, and commensal), based on clinical information and site of isolation. Twenty serotypes were identified, and the predominant serotypes were 1/2 and 7. Fifty-eight sequence types (STs) were identified, and the predominant ST was ST28. Associations among serotypes, STs, and pathotypes were investigated using odds ratio and clustering analyses. Evaluation of serotype and ST with pathotype identified a majority of isolates of serotypes 1, 1/2, 2, 7, 14, and 23 and ST1, ST13, ST25, ST28, ST29, ST94, ST108, ST117, ST225, ST373, ST961, and ST977 as associated with the pathogenic pathotype. Serotypes 21 and 31, ST750, and ST821 were associated with the commensal pathotype, which is composed of isolates from farms with no known history of S. suis-associated disease. Our study demonstrates the use of serotyping and MLST to differentiate pathogenic from commensal isolates and establish links between pathotype and subtype, thus increasing the knowledge about S. suis strains circulating in the United States.

Isolation and in vitro antimicrobial susceptibility of porcine Lawsonia intracellularis from Brazil and Thailand.

BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium which cannot be cultured by conventional bacteriological methods. Furthermore, L. intracellularis needs enriched medium and a unique atmosphere for isolation, cultivation and propagation. Because of this,there are only a few isolates of L. intracellularis available and few studies in vitro demonstrating the susceptibility of this bacterium to antimicrobial agents. The objectives of this study were to isolate South American and Southeast Asia strains of L.intracellularis and to determine the in vitro antimicrobial activity against these isolates. Tested antimicrobials included: chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin(against both Brazilian and Thailand strains) and additionally, amoxicillin, zinc-bacitracin, carbadox, enrofloxacin, gentamicin, sulfamethazine, trimethoprim, spectinomycin and a combination (1:1) of spectinomycin and lincomycin were also tested against the Thai isolates. The minimum inhibitory concentration (MIC) was determined by the antimicrobial activity that inhibited 99% of L. intracellularis growth in a cell culture as compared to the control (antimicrobial-free). RESULTS: Two strains from Brazil and three strains from Thailand were successfully isolated and established in cell culture. Each antimicrobial was evaluated for intracellular and extracellular activity. Pleuromutilin group (valnemulin and tiamulin) and carbadox were the most active against L. intracellularis strains tested. Tylosin showed intermediate activity, chlortetracycline had variable results between low and intermediate activity, as well as spectinomycin, spectinomycin and lincomycin, amoxicillin, sulfamethazine and enrofloxacin. L. intracellularis was resistant to lincomycin, gentamicin, trimethoprim, colistin and bacitracin in in vitro conditions. CONCLUSIONS: This is the first report of isolation of L. intracellularis strains from South America and Southeast Asia and characterization of the antimicrobial susceptibility patterns of these new strains.

Evaluation of mouse enteroids as a model for Lawsonia intracellularis infection.

Lawsonia intracellularis, an obligate intracellular bacterium, is an important enteric pathogen in pig herds and horse farms worldwide. The hallmark feature of L. intracellularis infection is the proliferation of epithelial cells in intestinal crypts. A major limitation to the study of L. intracellularis infection is the lack of an in vitro model that reproduces the changes observed in proliferative enteropathy. Here we investigated the suitability of mouse enteroids as a model to study L. intracellularis infection. Mouse enteroids were microinjected with L. intracellularis, filter-sterilized L. intracellularis culture supernatant, or sterile cell culture media (DMEM). L. intracellularis antigen was detected in mouse enteroids by immunohistochemistry and was located mostly in the basal region of the epithelium. There was no differential growth of enteroids among treatment groups, and cellular proliferation was not increased in L. intracellularis-infected enteroids in relation to non-infected enteroids based on immunofluorescence staining. L. intracellularis infection did not induce changes in gene expression of Ki-67 (proliferation marker), Sox9 (marker for transit amplifying cells) and Muc2 (marker for goblet cells). These results indicate that although L. intracellularis antigen is detectable in mouse enteroids, indicating susceptibility to infection, mouse enteroids fail to replicate the cellular proliferation and gene expression changes observed in proliferative enteropathy. Nevertheless, we have successfully demonstrated that mouse enteroids can be used to model days-long intracellular pathogen infection, serving as potential models for the study of other pathogens of interest in veterinary medicine.

The effects of zinc amino acid complex supplementation on the porcine host response to Lawsonia intracellularis infection.

Lawsonia intracellularis is among the most important enteric pathogens of swine and antibiotic alternatives are needed to help mitigate the negative effects of infection. Zinc is an essential trace mineral known to be crucial for maintaining intestinal barrier function and proper immune response. In this study, we investigated the porcine host response to L. intracellularis infection when supplemented with a zinc-amino acid complex, a form of zinc that can lead to greater bioavailability when compared to traditional inorganic forms of zinc. Our results show that a zinc-amino acid complex supplementation with a final concentration of 125 ppm of zinc in feed significantly (p < 0.05) decreased the number of animals with lesions and severity of lesions caused by L. intracellularis. Animals supplemented with the zinc-amino acid complex also exhibited a significantly (p < 0.05) earlier onset of seroconversion as well as an increased number of T cells in infected and non-infected intestinal tissue. This study demonstrated that this zinc-amino acid complex aids the host in responding to L. intracellularis infection and may be a new approach to help minimize negative effects of disease.

Lawsonia intracellularis in Pigs: Progression of Lesions and Involvement of Apoptosis.

The purpose of this study was to follow the progression of gross and histologic lesions and apoptosis events in Lawsonia intracellularis-infected enterocytes through the course of the disease, proliferative enteropathy (PE). Thirty 5-week-old pigs were divided into 2 groups: 20 challenged and 10 control animals. Groups of 3 pigs, 2 challenged and 1 control, were euthanized at 1, 3, 5, 8, 11, 15, 19, 24, 29, and 35 days after inoculation. Complete necropsies were performed with gross evaluation. Tissue samples from different sites of the gastrointestinal tract and other visceral organs were collected for routine histologic staining and for immunohistochemistry (IHC) for L. intracellularis. In addition, caspase-3, terminal deoxyuridine nick-end labeling assay, and electron microscopy were performed in ileum samples. Macroscopic and histologic lesions suggestive of PE were first detected 11 days after infection and continued through day 24. L. intracellularis antigen was first detected in the intestine by IHC on day 5 after inoculation, and the bacterium was first detected by transmission electron microscopy on day 15. Positive IHC staining for [L. intracellularis] and enterocyte proliferation, but no gross lesion, were detected on day 29. All 3 pigs euthanized on day 35 were grossly and histologically normal and IHC negative. Hyperplastic crypts in challenge pigs had more apoptotic cells on days 15, 19, and 24 postinfection ( P < .05) compared to control pigs. Our results demonstrated the progression of lesions and infection by L. intracellularis and that inhibition of enterocyte apoptosis is not involved in the pathogenesis of proliferative enteropathy.

Antimicrobial Susceptibility Patterns of Brachyspira Species Isolated from Swine Herds in the United States.

Outbreaks of swine dysentery, caused by Brachyspira hyodysenteriae and the recently discovered "Brachyspira hampsonii," have reoccurred in North American swine herds since the late 2000s. Additionally, multiple Brachyspira species have been increasingly isolated by North American diagnostic laboratories. In Europe, the reliance on antimicrobial therapy for control of swine dysentery has been followed by reports of antimicrobial resistance over time. The objectives of our study were to determine the antimicrobial susceptibility trends of four Brachyspira species originating from U.S. swine herds and to investigate their associations with the bacterial species, genotypes, and epidemiological origins of the isolates. We evaluated the susceptibility of B. hyodysenteriae, B. hampsonii, Brachyspira pilosicoli, and Brachyspira murdochii to tiamulin, valnemulin, doxycycline, lincomycin, and tylosin by broth microdilution and that to carbadox by agar dilution. In general, Brachyspira species showed high susceptibility to tiamulin, valnemulin, and carbadox, heterogeneous susceptibility to doxycycline, and low susceptibility to lincomycin and tylosin. A trend of decreasing antimicrobial susceptibility by species was observed (B. hampsonii > B. hyodysenteriae > B. murdochii > B. pilosicoli). In general, Brachyspira isolates from the United States were more susceptible to these antimicrobials than were isolates from other countries. Decreased antimicrobial susceptibility was associated with the genotype, stage of production, and production system from which the isolate originated, which highlights the roles of biosecurity and husbandry in disease prevention and control. Finally, this study also highlights the urgent need for Clinical and Laboratory Standards Institute-approved clinical breakpoints for Brachyspira species, to facilitate informed therapeutic and control strategies.

Characterization and Recognition of Brachyspira hampsonii sp. nov., a Novel Intestinal Spirochete That Is Pathogenic to Pigs.

Swine dysentery (SD) is a mucohemorrhagic colitis of swine classically caused by infection with the intestinal spirochete Brachyspira hyodysenteriae Since around 2007, cases of SD have occurred in North America associated with a different strongly beta-hemolytic spirochete that has been molecularly and phenotypically characterized and provisionally named "Brachyspira hampsonii." Despite increasing international interest, B. hampsonii is currently not recognized as a valid species. To support its recognition, we sequenced the genomes of strains NSH-16T, NSH-24, and P280/1, representing B. hampsonii genetic groups I, II, and III, respectively, and compared them with genomes of other valid Brachyspira species. The draft genome of strain NSH-16T has a DNA G+C content of 27.4% and an approximate size of 3.2 Mb. Genomic indices, including digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI), clearly differentiated B. hampsonii from other recognized Brachyspira species. Although discriminated genotypically, the three genetic groups are phenotypically similar. By electron microscopy, cells of different strains of B. hampsonii measure 5 to 10 µm by 0.28 to 0.34 µm, with one or two flat curves, and have 10 to 14 periplasmic flagella inserted at each cell end. Using a comprehensive evaluation of genotypic (gene comparisons and multilocus sequence typing and analysis), genomic (dDDH, ANI, and AAI) and phenotypic (hemolysis, biochemical profiles, protein spectra, antibiogram, and pathogenicity) properties, we classify Brachyspira hampsonii sp. nov. as a unique species with genetically diverse yet phenotypically similar genomovars (I, II, and III). We designate the type strain NSH-16 (= ATCC BAA-2463 = NCTC 13792).

Molecular Epidemiology of Novel Pathogen "Brachyspira hampsonii" Reveals Relationships between Diverse Genetic Groups, Regions, Host Species, and Other Pathogenic and Commensal Brachyspira Species.

Outbreaks of bloody diarrhea in swine herds in the late 2000s signaled the reemergence of an economically significant disease, swine dysentery, in the United States. Investigations confirmed the emergence of a novel spirochete in swine, provisionally designated "Brachyspira hampsonii," with two genetically distinct clades. Although it has since been detected in swine and migratory birds in Europe and North America, little is known about its genetic diversity or its relationships with other Brachyspira species. This study characterizes B. hampsonii using a newly developed multilocus sequence typing (MLST) approach and elucidates the diversity, distribution, population structure, and genetic relationships of this pathogen from diverse epidemiological sources globally. Genetic characterization of 81 B. hampsonii isolates, originating from six countries, with our newly established MLST scheme identified a total of 20 sequence types (STs) belonging to three clonal complexes (CCs). B. hampsonii showed a heterogeneous population structure with evidence of microevolution locally in swine production systems, while its clustering patterns showed associations with its epidemiological origins (country, swine production system, and host species). The close genetic relatedness of B. hampsonii isolates from different countries and host species highlights the importance of strict biosecurity control measures. A comparative analysis of 430 isolates representing seven Brachyspira species (pathogens and commensals) from 19 countries and 10 host species depicted clustering by microbial species. It revealed the close genetic relatedness of B. hampsonii with commensal Brachyspira species and also provided support for the two clades of B. hampsonii to be considered a single species.

FATAL SUPPURATIVE INFLAMMATION IN DAURIAN PIKAS (OCHOTONA DAUURICA) NATURALLY INFECTED WITH CASTELLANIELLA GINSENGISOLI-LIKE BACTERIA.

Fifteen Daurian pika (Ochotona dauurica) were introduced to a zoological collection in December 2011 as founders for a planned breeding colony. Despite breeding success, the colony shrunk over 37 mo to 11 animals. Mortality in 11 of 46 deceased animals, including wild-caught "founders" and captive-born offspring, was associated with a suppurative inflammation and abscess formation of the spleen, skin, peripheral and internal lymph nodes, liver, lungs, kidney, or a combination of organs. Gram-negative, non-fermenting, rod-shaped bacteria were isolated from the abscesses in all cases. Steiner Chapman silver stain identified rod-shaped bacteria in the abscesses of seven animals. The bacteria were not detected with Gram stain, acid-fast stain, or Grocott silver methenamine stain and was not detectable by periodic acid-Schiff reaction. In two cases, including the index case, the bacteria were presumptively identified as Ralstonia pickettii on the basis of conventional biochemical characterization. The bacteria in the other cases were not further classifiable with conventional methods. Matrix-assisted laser desorption/ionization time of flight mass spectrometry and 16s rDNA gene sequencing resulted in identification to the genus level as Castellaniella in 10 of 12 cases. Comparative 16S rDNA gene sequence analysis showed that these isolates and Castellaniella ginsengisoli Strain DCY36T were 99% similar. Castellaniella ginsengisoli, a gram-negative bacterium isolated from soil of a ginseng field in South Korea, has not previously been associated with disease in animals or humans. It is uncertain how the bacterium was introduced to the Daurian pika colony or how it spread.

Laser microdissection coupled with RNA-seq analysis of porcine enterocytes infected with an obligate intracellular pathogen (Lawsonia intracellularis).

BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium and the etiologic agent of proliferative enteropathy. The disease is endemic in pigs, emerging in horses and has been described in various other species including nonhuman primates. Cell proliferation is associated with bacterial replication in enterocyte cytoplasm, but the molecular basis of the host-pathogen interaction is unknown. We used laser capture microdissection coupled with RNA-seq technology to characterize the transcriptional responses of infected enterocytes and the host-pathogen interaction. RESULTS: Proliferative enterocytes was associated with activation of transcription, protein biosynthesis and genes acting on the G1 phase of the host cell cycle (Rho family). The lack of differentiation in infected enterocytes was demonstrated by the repression of membrane transporters related to nutrient acquisition. The activation of the copper uptake transporter by infected enterocytes was associated with high expression of the Zn/Cu superoxide dismutase by L. intracellularis. This suggests that the intracellular bacteria incorporate intracytoplasmic copper and express a sophisticated mechanism to cope with oxidative stress. CONCLUSIONS: The feasibility of coupling microdissection and RNA-seq was demonstrated by characterizing the host-bacterial interactions from a specific cell type in a heterogeneous tissue. High expression of L. intracellularis genes encoding hypothetical proteins and activation of host Rho genes infers the role of unrecognized bacterial cyclomodulins in the pathogenesis of proliferative enteropathy.

Comparative genome sequencing identifies a prophage-associated genomic island linked to host adaptation of Lawsonia intracellularis infections.

Lawsonia intracellularis is an obligate intracellular bacterium and the causative agent of proliferative enteropathy (PE). The disease is endemic in pigs, emerging in horses and has also been reported in a variety of other animal species, including nonhuman primates. Comparing the whole genome sequences of a homologous porcine L. intracellularis isolate cultivated for 10 and 60 passages in vitro, we identified a 18-kb prophage-associated genomic island in the passage 10 (pathogenic variant) that was lost in the passage 60 (non-pathogenic variant). This chromosomal island comprises 15 genes downstream from the prophage DLP12 integrase gene. The prevalence of this genetic element was evaluated in 12 other L. intracellularis isolates and in 53 infected animals and was found to be conserved in all porcine isolates cultivated for up to 20 passages and was lost in isolates cultivated for more than 40 passages. Furthermore, the prophage region was also present in 26 fecal samples derived from pigs clinically affected with both acute and chronic forms of the disease. Nevertheless, equine L. intracellularis isolates evaluated did not harbor this genomic island regardless of the passage in vitro. Additionally, fecal samples from 21 clinically affected horses and four wild rabbits trapped in horse farms experiencing PE outbreaks did not show this prophage-associated island. Although the presence of this prophage-associated island was not essential for a virulent L. intracellularis phenotype, this genetic element was porcine isolate-specific and potentially contributed to the ecological specialization of this organism for the swine host.

Evaluation of the role of clathrin and bacterial viability in the endocytosis of Lawsonia intracellularis.

Lawsonia intracellularis is an obligate intracellular bacterium and causative agent of proliferative enteropathy. The pathogenesis of L. intracellularis is not completely understood, including the endocytic mechanisms to access the host cell cytoplasm. In this study, we evaluated the mechanisms involved in endocytosis of L. intracellularis in vitro using intestinal porcine epithelial cells (IPEC-J2). Confocal microscopy was used to co-localize L. intracellularis and clathrin. Clathrin gene knockdown was then applied to verify whether L. intracellularis endocytosis is clathrin-dependent. Finally, internalization of viable and non-viable (bacteria were inactivated by heat) L. intracellularis organisms were assessed to study the role of the host cell during bacterial endocytosis. L. intracellularis organisms were observed co-localized with clathrin by confocal microscopy but the amount of L. intracellularis internalized in cells, with and without clathrin knockdown, did not differ statistically. The internalization of non-viable L. intracellularis showed a decrease in the internalization in cells with less clathrin synthesis (P<0.05). The present study is the first to elucidate the involvement of clathrin in the endocytosis of L. intracellularis. Clathrin-mediated endocytosis was shown to be an important, but not required, process for L. intracellularis internalization in porcine intestinal epithelial cells. Independence of bacterial viability for host cell internalization was also confirmed.

An alternative method for cultivation of Lawsonia intracellularis.

An alternative method for the cultivation of Lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, was developed using an Original Space Bag inflated with a mixture of gas containing 10% hydrogen, 10% carbon dioxide, and 80% nitrogen. The flexibility of this protocol allows the testing of various environmental conditions for static cultivation of this bacterium and the development of diagnostic techniques.

Evidence of host adaptation in Lawsonia intracellularis infections.

BACKGROUND: Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate. MATERIALS AND METHODS: Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (109L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response. RESULTS: Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate. CONCLUSIONS: Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated L. intracellularis strain.

Evaluation of the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) system in the detection of mastitis pathogens from bovine milk samples.

MALDI-TOF is a chemistry analytical tool that has recently been deployed in the identification of microorganisms isolated from nosocomial environments. Its use in diagnostics has been extremely advantageous in terms of cost effectiveness, sample preparation easiness, turn-around time and result analysis accessibility. In the dairy industry, where mastitis causes great financial losses, a rapid diagnostic method such as MALDI-TOF could assist in the control and prevention program of mastitis, in addition to the sanitation and safety level of the dairy farms and processing facility. However, the diagnostic strengths and limitations of this test method require further understanding. In the present study, we prospectively compared MALDI-TOF MS to conventional 16S rDNA sequencing method for the identification of pathogens recovered from milk associated with clinical and subclinical bovine mastitis cases. Initially, 810 bacterial isolates were collected from raw milk samples over a period of three months. However, only the isolates (481) having both 16S rDNA sequencing and MALDI-TOF identification were included in the final phase of the study. Among the 481 milk isolates, a total of 26 genera (12 g-postive and 14 g-negative), including 71 different species, were taxonomically charecterized by 16S rDNA at the species level. Comparatively, MALDI-TOF identified 17 genera (9 g-positive and 8 g-negative) and 33 differernt species. Overall, 445 (93%) were putatively identified to the genus level by MALDI-TOF MS and 355 (74%) were identified to the species level, but no reliable identification was obtained for 16 (3.3%), and 20 (4.2%) discordant results were identified. Future studies may help to overcome the limitations of the MALDI database and additional sample preparation steps might help to reduce the number of discordances in identification. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional identification methods for common mastitis pathogens, routinely isolated from raw milk. Thus it's adoption will strengthen the capacity, quality, and possibly the scope of diagnostic services to support the dairy industry.

Proposed virulence-associated genes of Streptococcus suis isolates from the United States serve as predictors of pathogenicity.

BACKGROUND: There is limited information on the distribution of virulence-associated genes (VAGs) in U.S. Streptococcus suis isolates, resulting in little understanding of the pathogenic potential of these isolates. This lack also reduces our understanding of the epidemiology associated with S. suis in the United States and thus affects the efficiency of control and prevention strategies. In this study we applied whole genome sequencing (WGS)-based approaches for the characterization of S. suis and identification of VAGs. RESULTS: Of 208 S. suis isolates classified as pathogenic, possibly opportunistic, and commensal pathotypes, the genotype based on the classical VAGs (epf, mrp, and sly encoding the extracellular protein factor, muramidase-release protein, and suilysin, respectively) was identified in 9% (epf+/mrp+/sly+) of the pathogenic pathotype. Using the chi-square test and LASSO regression model, the VAGs ofs (encoding the serum opacity factor) and srtF (encoding sortase F) were selected out of 71 published VAGs as having a significant association with pathotype, and both genes were found in 95% of the pathogenic pathotype. The ofs+/srtF+ genotype was also present in 74% of 'pathogenic' isolates from a separate validation set of isolates. Pan-genome clustering resulted in the differentiation of a group of isolates from five swine production companies into clusters corresponding to clonal complex (CC) and virulence-associated (VA) genotypes. The same CC-VA genotype patterns were identified in multiple production companies, suggesting a lack of association between production company, CC, or VA genotype. CONCLUSIONS: The proposed ofs and srtF genes were stronger predictors for differentiating pathogenic and commensal S. suis isolates compared to the classical VAGs in two sets of U.S. isolates. Pan-genome analysis in combination with metadata (serotype, ST/CC, VA genotype) was illustrated to be a valuable subtyping tool to describe the genetic diversity of S. suis.

Comparison of feces versus rectal swabs for the molecular detection of Lawsonia intracellularis in foals with equine proliferative enteropathy.

The purpose of the current study was to compare the molecular detection rate of Lawsonia intracellularis between feces and rectal swabs collected from 42 foals with suspected equine proliferative enteropathy (EPE). Fecal samples and rectal swabs were processed for DNA purification by using an automated extraction system. The purified DNA was then analyzed by real-time polymerase chain reaction (PCR) for the presence of the aspartate ammonia lyase (aspA) gene of L. intracellularis. Absolute quantitation was calculated by using a standard curve for L. intracellularis and expressed as copy numbers of the aspA gene of L. intracellularis per microliter of purified DNA. The combined PCR detection rate for L. intracellularis was 90%, with 38 foals testing PCR positive in feces (33 samples), rectal swabs (32), or both (27). Six foals tested PCR positive only in feces, whereas 5 tested positive only in rectal swabs. Feces yielded a significantly higher aspA gene copy number of L. intracellularis than rectal swabs. Feces and rectal swabs tested PCR negative from 4 foals. In conclusion, the results showed that feces yielded similar numbers of PCR-positive results, with a higher L. intracellularis aspA gene load than rectal swabs. By analyzing dual samples, the PCR detection rate for L. intracellularis increased from 76% and 79% for rectal swabs and feces, respectively, to 90%. Rectal swabs should be considered as an alternative sample type for EPE-suspected patients with decreased or no fecal output.

Development of a polymerase chain reaction assay for quantification of Lawsonia intracellularis.

The objective of the present study was to develop a quantitative polymerase chain reaction (qPCR) assay using SYBR Green for quantification of Lawsonia intracellularis in cell culture and pig fecal samples. Specific primers were designed and tested using the aspartate ammonia-lyase (aspA) gene as a target. Serial 10-fold dilutions of cell culture samples and several sets of spiked feces were used for qPCR optimization. The lower limit of the linear range of the assay in cell culture was 5.1 x 10(2) L. intracellularis/ml. A concentration of between 2.55 x 10(4) and 2.55 x 10(3) L. intracellularis/g was the lower limit of the linear range when testing community DNA from spiked fecal samples. From both cell culture and fecal samples, L. intracellularis could be detected but not accurately quantified at levels approximately 1 log below the linear range. No cross-reactivity of qPCR was found when the assay was tested using the DNA extracted from 16 species of enteric bacteria commonly found in pig feces or closely related to L. intracellularis. The new qPCR assay might prove to be a sensitive, specific, precise, and accurate method for the detection and quantification of L. intracellularis in field samples.

Evaluation of swine enteroids as in vitro models for Lawsonia intracellularis infection1,2.

The enteric pathogen Lawsonia intracellularis is one of the main causes of diarrhea and compromised weight gain in pigs worldwide. Traditional cell-line cultures have been used to study L. intracellularis pathogenesis. However, these systems fail to reproduce the epithelial changes observed in the intestines of L. intracellularis-infected pigs, specifically, the changes in intestinal cell constitution and gene expression. A more physiologically accurate and state-of-the-art model is provided by swine enteroids derived from stem cell-containing crypts from healthy pigs. The objective of this study was to verify the feasibility of two-dimensional swine enteroids as in vitro models for L. intracellularis infection. We established both three- and two-dimensional swine enteroid cultures derived from intestinal crypts. The two-dimensional swine enteroids were infected by L. intracellularis in four independent experiments. Enteroid-infected samples were collected 3 and 7 d postinfection for analysis using real-time quantitative PCR and L. intracellularis immunohistochemistry. In this study, we show that L. intracellularis is capable of infecting and replicating intracellularly in two-dimensional swine enteroids derived from ileum.